Methods for diagnosing and treating neuroendocrine cancer

ABSTRACT

The present invention relates to a method for diagnosing neuroendocrine cancers via detecting the presence of N-methyl D-asparate-associated (NMDA) glutamate receptors type 1 and/or type 2. Methods for preventing and treating neuroendocrine cancers are also disclosed.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/875,067, filed Oct. 19, 2007 now abandoned,which is a continuation-in-part application of PCT/US2006/014660, filedApr. 19, 2006, which claims benefit of U.S. Provisional PatentApplication Ser. No. 60/672,829, filed Apr. 19, 2005, the contents ofwhich are incorporated herein by reference in their entirety.

This invention was made with government support under Grant No. CA19613awarded by the National Cancer Institute and Grant No. DAM D17-94-J-4288awarded by the Department of Defense. Therefore, the U.S. government hascertain rights in the invention.

INTRODUCTION Background of the Invention

Glutamate is the major excitatory neurotransmitter in central nervoussystem (CNS) and as such, the glutamate receptors play a vital role inthe mediation of excitatory synaptic transmission. The ionotropicreceptors themselves are ligand-gated ion channels, i.e., on bindingglutamate that has been released from a companion cell, charged ionssuch as Na⁺ and Ca²⁺ pass through the receptor complex therebydepolarizing the plasma membrane and generating an electrical current.

The ionotropic glutamate receptors are multimeric assemblies of four orfive subunits, and are subdivided into three groups (AMPA, NMDA andKainate receptors) based on their pharmacology structural similarities.All ionotropic glutamate receptor subunits share a common basicstructure. Like other ligand-gated ion channels, such as the GABA_(A)receptor, the ionotropic glutamate receptor subunits possess fourhydrophobic regions within the central portion of the sequence(transmembrane I-IV). However, in contrast to other receptor subunits,the transmembrane II domain forms a re-entrant loop giving thesereceptor subunits an extracellular N-terminus and intracellularC-terminus. In addition, the long loop between transmembrane III andtransmembrane IV, which is intracellular in other ligand-gated ionchannel subunits, is exposed to the cell surface, and forms part of thebinding domain with the C-terminal half of the N-terminus.

NMDA receptors are composed of assemblies of NMDA Type 1 (NMDAR1) andNMDA Type 2 (NMDAR2) glutamate receptors, which can be one of fourseparate gene products (NMDAR2 a-d). Expression of both subunits isrequired to form functional channels. The glutamate binding domain isformed at the junction of NMDAR1 and NMDAR2. In addition to glutamate,the NMDA receptor requires a co-agonist, glycine, to bind to allow thereceptor to optimally function. The glycine binding site is found on theNMDAR2 and NMDAR2b also possesses a binding site for polyamines,regulatory molecules that modulate the functioning of the NMDA receptor.

North et al. ((1997) Mol. Chem. Neuropathol. 30(1-2):77-94) teach theexpression of structurally normal and functional NMDA receptors byacetylcholine-producing human LA-N-2 neuroblastoma cells in culture.Cell cytotoxicity was shown by a neutral red cytotoxicity assay to beincreased through incubation with glutamate at 1 and 10 mM by 27 and37%, and through incubation with NPG at 0.1 and 1 mM by 28 and 46%.Further, a voltage-dependent tetrodotoxin-sensitive inward sodiumcurrent was found to be increased (×1.5) by L-glutamic acid and by ACDAand NPG NMDA agonists in the presence of glycine. It was concluded thatthe glutamate activities observed in human LA-N2 neuroblastoma cellsappeared to occur through the activation of functional NMDA receptors inmuch the same way as reported for neurons, and both glutamate and NMDAagonists can be toxic to these neuroblastoma cells.

Rzeski et al. ((2002) Biochem. Pharmacol. 64:1195-200) teach theinfluence of glutamate antagonists on the proliferation and migration oftumor cells. This reference teaches that glutamate N-methyl-D-aspartate(NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)antagonists inhibit the proliferation of human colon adenocarcinoma,astrocytoma, breast and non-small cell lung carcinoma, and neuroblastomacells in vitro. The antiproliferative effect of glutamate antagonists isCa⁺²-dependent and results from decreased cell division and increasedcell death. Glutamate antagonists produce morphological alterations intumor cells, which consist of reduced membrane ruffling and pseudopodialprotrusions, and decrease their motility and invasive growth.Furthermore, glutamate antagonists enhance in vitro cytostatic andcytotoxic effects of common chemotherapeutic agents used in cancertherapy.

Choi et al. ((2004) J. Oral Pathol. Med. 33(9):533-7) disclose thatimmunohistochemical staining for NMDAR1 was positive in 50 of 81 oralsquamous cell carcinoma (OSCC) cases, while it was negative in thecontrol. NMDAR1 expression was significantly associated with a lymphnode metastasis (P=0.008), the tumor size (P<0.001), and the cancerstage (P=0.034). The patients whose tumors expressed NMDAR1 had asignificantly poorer survival than the patients who wereNMDAR1-negative.

U.S. Pat. No. 6,797,692 teaches a method for treating cancer (e.g., acell exhibiting abnormal or uncontrolled cell growth with resultinginvasion and destruction of neighboring tissue). The method involvesadministering an inhibitor of the interaction between glutamate with aglutamate receptor complex (i.e., AMPA, KA or NMDA receptor complexes).

SUMMARY OF THE INVENTION

The present invention is a method for diagnosing a neuroendocrinecancer. The method involves the steps of isolating a sample from apatient and detecting the presence of at least one NMDA glutamatereceptor, or nucleic acids encoding the same, in said sample wherein thepresence of at least one NMDA glutamate receptor, or nucleic acidsencoding the same, is indicative of said patient having or at risk ofhaving a neuroendocrine cancer.

The present invention is also a method for decreasing proliferation of aneuroendocrine tumor cell. This method involves contacting aneuroendocrine tumor cell with an effective amount of an agent whichinhibits or decreases the activity or expression of an NMDA glutamatereceptor thereby decreasing proliferation of the neuroendocrine tumorcell.

The present invention is further a method for preventing or treating aneuroendocrine cancer in a subject. This method includes administeringto a subject having or at risk of having a neuroendocrine cancer aneffective amount of an agent which inhibits or decreases the activity orexpression of an NMDA glutamate receptor thereby preventing or treatinga neuroendocrine cancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of memantine on NCI-H345 and NCI-H82 cellproliferation. FIG. 1A shows the percentage reduction in cell viabilityproduced in NCI-H345 classical cells by the action of irreversibleNMDAR1 receptor antagonist memantine following 24, 48, and 72 hoursincubation. FIG. 1B shows the percentage reduction in cell viabilityproduced in NCI-H82 variant cells by the action of irreversible NMDAR1receptor antagonist memantine following 24, 48, and 72 hours incubation.Cell viability was reduced to about 10% of control and assessed usingAlamar Blue staining. Reductions in viability were highly significant(p<0.0001) and IC₅₀s were obtained with 80-100 μM doses of antagonist at48 and 72 hours of incubation.

FIG. 2 shows the effect of MK-801 on NCI-H345 and NCI-H82 cellproliferation. FIG. 2A shows the percentage reduction in cell viabilityproduced in NCI-H345 classical cells by the action of irreversibleNMDAR1 receptor antagonist MK-801 (dizocilpine maleate) following 24,48, and 72 hours of incubation. FIG. 2B shows the percentage reductionin cell viability produced in NCI-H82 variant cells by the action ofirreversible NMDAR1 receptor antagonist MK-801 following 24, 48, and 72hours incubation. Cell viability was reduced to about 10% of control andassessed using Alamar Blue staining. Reductions in viability were highlysignificant (p<0.0001) and IC₅₀s were obtained with ˜200 μM doses ofantagonist at 48 and 72 hours of incubation.

FIG. 3 shows the effect of Ifenprodil on NCI-H345 and NCI-H82 cellproliferation. FIG. 3A shows the percentage reduction in cell viabilityproduced in NCI-H345 classical cells by the action of irreversibleNMDAR2B receptor antagonist Ifenprodil following 24, 48, and 72 hoursincubation. FIG. 3B shows the percentage reduction in cell viabilityproduced in NCI-H82 variant cells by the action of irreversible NMDAR2Breceptor antagonist Ifenprodil following 24, 48, and 72 hoursincubation. Cell viability was reducted to about 10% of control andassessed using Alamar Blue staining. Reductions in viability were highlysignificant (p<0.0001) and IC₅₀s were obtained with 150-200 μM doses ofantagonist at 48 and 72 hours of incubation.

FIG. 4 shows the inhibition of SCLC NCI-H345 tumors in control (closedsymbol) and treated (open symbol) animals. Growth of small-cell tumorsfrom NCI-H345 cells was significantly (p<0.0001) reduced by treatmentwith escalating doses of MK-801 from 0.1 mg/kg once daily to 0.3 mg/kggiven twice daily as indicated. Tumor volume measured by micrometer wasexpressed as the product of length×width×breadth and was evaluated on adaily basis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of N-methylD-asparate-associated (NMDA) glutamate receptors for diagnosingneuroendocrine tumors as well as targets for the prevention or treatmentof neuroendocrine tumors. An NMDA glutamate receptor, as used in thecontext of the present invention, is intended to include NMDA Type 1(i.e., NMDAR1) and NMDA Type 2 (i.e., NMDAR2abcd) glutamate receptors.

As used herein, a neuroendocrine cancer or tumor is either one whicharises from the neuroendocrine system, a diffuse system in which thenervous system and the hormones of the endocrine glands interact, orfrom non-endocrine cells by acquiring some of the properties ofneuroendocrine cells through an oncogenic process such as SelectiveTumour gene Expression of Peptides essential for Survival (STEPS) (see,North (2000) Exper. Physiol. 85S:27S-40S). Most of the well-describedadult neuroendocrine tumors are distinctive and arise from a knownprimary site, including the carcinoid, pheochromocytoma, and Merkel'scell tumors. Carcinoid tumors can be benign or malignant. Carcinoidcancers include stomach, pancreas, colon, liver, lung, ovarian, breast,testicular, and cervical cancer. Neuroendocrine tumors of the lungs areclassified as small cell carcinoma. It is characterized by its origin inlarge central airways and histological composition of sheets of smallcells with scanty cytoplasm. Small cell carcinoma is very aggressive,metastazing early and often. Pheochromocytoma is a cancer of the adrenalmedulla. This condition often causes the adrenal glands to make too muchcatecholamine. Pheochromocytoma may arise as part of a condition calledmultiple endocrine neoplasia (MEN) syndrome, which can result in othercancers of the endocrine system and hormonal abnormalities. Merkel'scell tumors are cancers that form on or just beneath the skin, butsometimes are also thought to arise from underlying soft tissue. Theyare also known as neuroendocrine cancer of the skin. Merkel's celltumors are fast-growing and often spread to other parts of the body. Inparticular embodiments of the present invention, a neuroendocrine canceris a carcinoid cancer such as breast cancer or SCLC.

It has now been found that neuroendocrine tumor cells express bothNMDAR1 and NMDAR2 glutamate receptors, whereas normal cells from thesesame tissues lack NMDAR1 and NMDAR2 expression. Analysis of theexpression of the NMDA receptors by cultured SCLC cells and tumortissues was carried out using RT-PCR of poly(A⁺) RNA preparations. Allfour SCLC cell lines tested (NCI-H345, DMS-53, NCI-H146 and NCI-H82)yielded in each case, a single overlapping product of size predictedfrom the structure of cDNA for human NMDAR1 from brain tissue, andreported for human LA-N-2 neuroblastoma cells (North, et al. (1997) Mol.Chem. Neuropath. 30:77-94). Cloning and nucleotide sequence analysis ofthese NMDA glutamate receptor RT-PCR products (488 bp and 263 bp),coding for portions of the extracellular domain, showed them to haveexact sequence homology with position 208-695 of the brain andneuroblastoma receptor, and sequence identity in this portion of theNMDAR1 receptor for all four SCLC cell lines. The region in the mRNAexamined represents approximately 30% of the open reading frame for theextracellular N-terminal domain for this receptor sub-unit. As was foundfor the mRNA from LA-N-2 cells, there was no evidence for alternatesplicing of the message as has been reported for NMDAR1 from rat brain(Moriyoshi, et al. (1991) Nature 354:31-37). RT-PCR of poly(A⁺) RNA wasunaffected by prior treatment of preparations with DNase, and noproducts were generated when initial treatment with RNase was performedor when reverse transcriptase was omitted from reaction mixtures.Nucleotide sequencing also revealed normal sequences for the two otherregions of NMDAR1 mRNA amplified giving predicted RT-PCR products of 300and 391 nucleotides. In addition, RT-PCR of NMDAR2B message providedpredicted products of 471 and 564 nucleotides, that upon nucleotidesequencing were shown to have sequences identical to those known in theart (Hess, et al. (1996) J. Pharmacol. Exper. Therap. 278:808-816).Likewise, real-time RT-PCR analysis of NMDAR2A indicated that thesmall-cell lung cancer cell lines NCI H345, NCI H146, and DMS 53 (allrepresenting primary disease) and NCI H82 (representing recurrentdisease) expressed this receptor.

There is a consensus that the molecular weight of the NMDAR1 proteinsubunit is 116 kDaltons, and a major band of approximately this size wasdisplayed for NCI-H345, NCI-H82, NCI-146, and DMS-53 cells in westernanalysis using affinity-purified PANN1 antibodies. A second minor bandof approximately 55 kD was also apparent. This second band could be anN-terminal break-down product of the receptor, because PANN1 is directedagainst an N-terminal moiety of the protein. Alternatively, the NMDAR2subunits are 165-180 kDaltons, and a major band of approximately 170kDaltons (in addition to bands corresponding to smaller proteins) wasdisplayed in western blots of all SCLC cell lines by the NMDAR2epolyclonal antibody employed (i.e., an antibody that recognizes theextracellular domain of NMDAR2B), indicating that NMDAR2A, B, or C ofnormal size is expressed by all of the cell lines. Further, the NMDAR2epolyclonal antibody indicated the presence of one or more of theseproteins in two breast cancer cell lines (i.e., MCF-7 and SKBR3).Indeed, RT-PCR analyses confirmed the presence of the NMDAR1 and NMDAR2Atranscripts in the MCF-7 and SKBr3 breast cancer cell lines. Coupledwith RT-PCR data, it was concluded that SCLC and breast cancer cellsexpress and translate NMDAR1 receptor as well as NMDAR2B and NMDAR2Aforms of the NMDAR2 receptor.

Immunohistochemical evaluation of SCLC tumor sections with anti-NMDAR1antibodies gave strong and clear positive staining for eight of tentumors, and weak, questionable staining in the other two cases examined.All of the 6 cases of metastatic and recurrent disease were among thosegiving a strong positive reaction. Normal cells in each section did notreact with the antibodies. No positive staining of normal lung and liverwas apparent with PANN1 anti-NMDAR1 antibodies for the same conditionsof IHC, except for Kufper cells in the liver and isolated macrophages inboth tissues. Similarly, immunohistochemical analysis of fixed breastcancer tissues indicated that breast cancer tissue expresses NMDAR1,whereas normal breast tissue epithelial or myoepithelial cells do notstain positive for the presence of this receptor.

Accordingly, the present invention is a method for diagnosing aneuroendocrine cancer in a patient suspected of having or at risk ofhaving a neuroendocrine tumor by detecting the presence of at least oneNMDA glutamate receptor. The method involves isolating a sample from thepatient and detecting the presence of at least one NMDA glutamatereceptor, or nucleic acids encoding the same, in the sample. In oneembodiment, the assay of the present invention is carried out bydetecting the presence of NMDAR1 or NMDAR2. In another embodiment, theassay of the present invention is carried out by detecting the presenceof both NMDAR1 and NMDAR2. In particular embodiments pertaining toNMDAR2, one or both of NMDAR2A and NMDAR2B are detected. As used in thecontext of the present invention, a sample is intended to mean anybodily fluid or tissue which is amenable to protein or nucleic acidanalysis. Suitable samples which can be analyzed in accordance with themethod of the present invention include, but are not limited to, sputum,vaginal or rectal swabs, biopsy samples, and the like isolated from apatient (e.g., human, livestock or companion animal) according tostandard clinical practices. It is contemplated that the sample can befrom an individual suspected of having a neuroendocrine tumor or from anindividual at risk of having a neuroendocrine tumor, e.g., an individualin remission or having a family history which predisposes the individualto neuroendocrine cancer.

In one embodiment of this diagnostic method, the presence of at leastone NMDA glutamate receptor is detected in assays using a binding agentwhich specifically binds to a NMDA glutamate receptor protein (i.e.,NDMAR1 and/or NMDAR2) and no other ionotropic glutamate receptor (e.g.,AMPA or Kainate receptors). In this embodiment, a sample is contactedwith a binding agent (e.g., antibody), which binds an NMDA glutamatereceptor, and the resulting receptor-binding agent complex is detectedusing standard assays (e.g., an immunoassay). When the binding agent is,for example, a peptide aptamer, the receptor-binding agent complex canbe directly detected by, for example, a detectable marker protein (e.g.,β-galactosidase, GFP or luciferase) fused to the aptamer. Subsequently,the presence or absence of the receptor-binding agent complex iscorrelated with the presence or absence, respectively, of aneuroendocrine tumor cells in the sample and therefore the presence orabsence, respectively, of a neuroendocrine cancer in the patient. It iscontemplated that one or more binding agents of the present inventioncan be used in conjunction with current non-invasive immuno-basedimaging techniques (e.g., mammography) to greatly enhance detection andearly diagnosis of neuroendocrine tumors. For example, as SCLC iscurrently diagnosed on the basis of gross morphological and histologicaldata obtained from biopsied tissue and is often identified after thedisease has reached its advanced stages, the instant method can be usedfor early diagnosis of SCLC. Additionally, ductal carcinoma in situ(DCIS) is often difficult to discern from atypical ductal hyperplasia(ADH), generally considered to be a benign affliction, on biopsiedtissue sections. These biopsied tissue samples could be stained usingbinding agents disclosed herein, allowing for differential diagnoses tobe made thereby improving subsequent treatment procedures and outcomes.

Binding agents for use in accordance with the instant invention includeantibodies, as well as peptide aptamers. In particular embodiments ofthe present invention, the binding agent specifically recognizes atleast a portion of the N-terminal extracellular domain of at least oneNMDA glutamate receptor (i.e., NMDAR1 or NMDAR2). The extracellulardomain of an NMDAR1 receptor can be found at about amino acid residues19-938 of GENBANK Accession number Q05586 (Planells-Cases, et al. (1993)Proc. Natl. Acad. Sci. USA 90:5057-5061). Likewise, the extracellulardomain of NMDAR2 receptors A, B, C, and D can be found at about aminoacid residues 23-28 to 555-583 of GENBANK Accession numbers NP_(—)000824(Monyer, et al. (1992) Science 256(5060):1217-21), NP_(—)000825 (Monyer,et al. (1992) supra), NP_(—)000826 Monyer, et al. (1992) supra) andNP_(—)000827 (Hess, et al. (1998) J. Neurochem. 70(3):1269-79),respectively. A particularly suitable antigenic fragment of theextracellular domain of NMDAR1 for generating antibodies to NMDAR1 isMet-Ser-Ile-Tyr-Ser-Asp-Lys-Ser-Ile-His (SEQ ID NO:5). Other suitableantigenic regions of the extracellular domain of these proteins can bereadily identified by the skilled artisan using any art-establishedcomputer algorithm for identifying such antigenic sequences (e.g.,Jamison and Wolf (1988) Bioinformatics 4:181-186; Carmenes, et al.(1989) Biochem Biophys Res Commun. 159(2):687-93). To generate a bindingagent specific for all isoforms of NMDAR2, it is desirable that anantigenic peptide common to all isoforms be used to generate the bindingagent. In one embodiment, the binding agent is specific for at least aportion of the extracellular domain of NMDAR2A, NMDAR2B, NMDAR2C, orNMDAR2D. In another embodiment, the binding agent is specific for atleast a portion of the extracellular domain of all isoforms of NMDAR2(i.e., A, B, C, and D).

Antibodies to an NMDA glutamate receptor can be generated using methodsthat are well-known in the art. Such antibodies can include, but are notbe limited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments, bispecific scFv fragments, Fd fragments and fragmentsproduced by a Fab expression library.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith an NMDA glutamate receptor protein or any fragment or oligopeptidethereof which has antigenic or immunogenic properties. An exemplaryfragment is the N-terminal extracellular domain of an NMDA glutamatereceptor (i.e., SEQ ID NO:5). Depending on the host species, variousadjuvants can be used to increase the immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areparticularly suitable.

An antibody to an NMDA glutamate receptor can be generated by immunizingan animal with an oligopeptide, peptide, or fragment, e.g., of theN-terminal extracellular domain of the NMDA glutamate receptor protein.Generally, such oligopeptides, peptides, or fragments have an amino acidsequence consisting of at least five amino acids and more desirably atleast 10 amino acids. Fragments of an NMDA glutamate receptor proteincan be generated by, for example, tryptic digestion and extraction froma preparative SDS-PAGE gel or by recombinant fragment expression andpurification. Further, short stretches of amino acids of an NMDAglutamate receptor antigen of the invention can be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule.

Monoclonal antibodies to an NMDA glutamate receptor protein of theinvention can be prepared using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique(Kohler, et al. (1975) Nature 256:495-497; Kozbor, et al. (1985) J.Immunol. Methods 81:31-42; Cote, et al. (1983) Proc. Natl. Acad. Sci.80:2026-2030; Cole, et al. (1984) Mol. Cell Biol. 62:109-120).

In addition, techniques developed for the production of humanized andchimeric antibodies, the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, et al. (1984) Proc.Natl. Acad. Sci. 81, 6851-6855; Neuberger, et al. (1984) Nature312:604-608; Takeda, et al. (1985) Nature 314:452-454). Alternatively,techniques described for the production of single chain antibodies canbe adapted, using methods known in the art, to produce specific singlechain antibodies. Antibodies with related specificity, but of distinctidiotypic composition, can be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton (1991) Proc. Natl. Acad.Sci. 88, 11120-11123).

Antibodies can also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as is well-known in the art(Orlandi, et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, etal. (1991) Nature 349:293-299).

Antibody fragments, which contain specific binding sites for an NMDAglutamate receptor protein, or a fragment thereof, can also begenerated. For example, such fragments include, but are not limited to,the F(ab′)₂ fragments which can be produced by pepsin digestion of theantibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, et al. (1989) Science 254:1275-1281).

Diabodies are also contemplated. A diabody refers to an engineeredantibody construct prepared by isolating the binding domains (both heavyand light chain) of a binding antibody, and supplying a linking moietywhich joins or operably links the heavy and light chains on the samepolypeptide chain thereby preserving the binding function (see, Holligeret al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak (1994)Structure 2:1121-1123). This forms, in essence, a radically abbreviatedantibody, having only the variable domain necessary for binding theantigen. By using a linker that is too short to allow pairing betweenthe two domains on the same chain, the domains are forced to pair withthe complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or diabodies,are bivalent and bispecific. It should be clear that any method togenerate diabodies, as for example described by Holliger, et al. (1993)supra, Poljak (1994) supra, Zhu, et al. (1996) Biotechnology 14:192-196,and U.S. Pat. No. 6,492,123, herein incorporated by reference, can beused.

Various immunoassays can be used for screening to identify antibodies,or fragments thereof, having the desired specificity for NMDA glutamatereceptor antigen. Numerous protocols for competitive binding (e.g,ELISA), latex agglutination assays, immunoradiometric assays, andkinetics (e.g., BIACORE™ analysis) using either polyclonal or monoclonalantibodies, or fragments thereof, are well-known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween a specific antibody and its cognate antigen. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes is suitable, but a competitive bindingassay can also be employed. Such assays can also be used in thedetection of an NMDA glutamate receptor in a sample.

Peptide aptamers which specifically bind to an NMDA glutamate receptorprotein can be rationally designed or screened for in a library ofaptamers (e.g., provided by Aptanomics SA, Lyon, France). In general,peptide aptamers are synthetic recognition molecules whose design isbased on the structure of antibodies. Peptide aptamers consist of avariable peptide loop attached at both ends to a protein scaffold. Thisdouble structural constraint greatly increases the binding affinity ofthe peptide aptamer to levels comparable to that of an antibody(nanomolar range). Likewise, aptamers which bind to nucleic acidsequences encoding an NMDA glutamate receptor protein, can also beidentified in library screens.

A binding agent can also be subjected to other biological activityassays, e.g., cancer cell growth assays, in order to evaluate itspotency or pharmacological activity and potential efficacy as atherapeutic agent. Such assays are described herein and are well-knownin the art.

In an alternate embodiment of the diagnostic method of the presentinvention, the presence of the NMDA glutamate receptor protein in asample (e.g., samples provided supra) is detected via the presence ofnucleic acid sequences encoding an NMDA glutamate receptor. Nucleic acidsequences encoding an NMDA glutamate receptor can be detected using anywell-known method including, but not limited to, northern blot analysis,reverse-transcriptase PCR, PCR, microarray, and the like. Due to theease of use, it is generally desirable to detect the nucleic acidsequences using a PCR-based approach. In general, this involvescontacting the sample with two or more PCR primers which specificallyhybridize with nucleic acids encoding an NMDA glutamate receptor orwhich flank the coding region of an NMDA glutamate receptor, subjectingthe sample to multiple steps of PCR amplification and detecting thepresence or absence of the amplified sequence (e.g., using gel analysis,blotting methods, or fluorescently-labeled primers). Alternatively, anoligonucleotide, an aptamer, a cDNA, an antibody, or a fragment thereof,which interacts with at least a portion of the nucleic acid encoding anNMDA glutamate receptor protein is configured in an array on a chip orwafer and used for detecting nucleic acids encoding an NMDA glutamatereceptor. Briefly, these techniques involve methods for analyzing largenumbers of genes rapidly and accurately. By tagging genes witholigonucleotides or using fixed probe arrays, one can employ chiptechnology to segregate target molecules as high density arrays andscreen these molecules on the basis of hybridization (see, e.g., Pease,et al. (1994) Proc. Natl. Acad. Sci. USA 91(11):5022-6; Fodor, et al.(1991) Science 251(4995):767-73).

Primers or oligonucleotides for use in this embodiment can be selectedfrom any region of the locus encoding an NMDA glutamate receptor proteinand generally specifically anneal and amplify at least a portion of anucleic acid encoding an NMDA glutamate receptor and no other nucleicacid encoding a closely related protein. Suitable primers foramplification of nucleic acids encoding NMDAR1 include those exemplifiedherein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) orcan be selected by the skilled artisan from the nucleic acid sequencesencoding NMDAR1 (i.e., GRIN1) disclosed in Foldes, et al. (1993) Gene131:293-298); Zimmer, et al. (1995) Gene 159:219-223), or Foldes, et al.(1994) Gene 147:303-304. See also GENBANK Accession Nos. NM_(—)000832,NM_(—)021569, and NM_(—)007327. Suitable primers for amplification ofnucleic acids encoding NMDAR2A-D can likewise be selected by the skilledartisan from the nucleic acid sequences encoding NMDAR2A-D (i.e.,GRIN2A-D) disclosed in Monyer, et al. (1992) supra or Hess, et al.(1998) supra. See also GENBANK Accession Nos. NM_(—)000833,NM_(—)000834, NM_(—)000835, and NM_(—)000836. Exemplary primers forNMDAR2B are set forth herein in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,and SEQ ID NO:9, whereas exemplary primers for NMDAR2A are set forth inSEQ ID NO:10 and SEQ ID NO:11.

In general, suitable primers are 12 to 30 bp in length and generate aPCR amplicon of 50, 100, 200 400, 600, 1000 bp or more in length. Inaccordance with this method, a geometrically amplified product isobtained only when the first and second nucleotide sequences occurwithin the same nucleic acid molecule encoding the NMDR glutamatereceptor protein. The fundamentals of non-degenerate PCR are well-knownto the skilled artisan, see, e.g. McPherson, et al., PCR, A PracticalApproach, IRL Press, Oxford, Eng. (1991).

Typically, the diagnostic method of the present invention will include apositive and/or negative control to assess the accuracy of the method.

In conjunction with the diagnostic method of the present invention, akit for identifying the presence of an NMDA glutamate receptor proteinis also provided. A kit of the invention includes a container containingat least one binding agent (e.g., an antibody) which specifically bindsa NMDA glutamate receptor protein. Alternatively, the kit containssuitable primers (e.g., those disclosed herein) for amplifying nucleicacids encoding at least one NMDA glutamate receptor. The kit can alsocontain other solutions necessary or convenient for carrying out theinvention. The container can be made of glass, plastic or foil and canbe a vial, bottle, pouch, tube, bag, etc. The kit may also containwritten information, such as procedures for carrying out the presentinvention or analytical information, such as the amount of reagentcontained in the first container means. The container can be in anothercontainer, e.g., a box or a bag, along with the written information.

In addition to being present, the NMDAR1 and NMDAR2 glutamate receptorswere found to be fully functional in the breast cancer cell lines.Growth and NMDA glutamate receptor activity of these cells were found torespond to the effects of glutamate (NMDAR1 agonist) in the presence ofglycine (NMDAR2 agonist) over 24 hours. Glutamate decreased cell growthin LAN2 cells, and increased proliferation in breast cancer cells underthe conditions used (Table 1, p<0.05).

TABLE 1 Fluorescence (nm) Cell Line Vehicle Agonist LAN2 465 420 MCF7485 510 SKBr3 345 445 Results represent triplicate studies performedwith alamar blue on LAN2 neuroblastoma, MCF-7 breast cancer, and SKBr3breast cancer cells examining the growth influence of 1 mM glutamate inthe presence of glycine over 24 hours.

Treatment of these cells with magnesium (NMDA1 antagonist) in thepresence of glycine (NMDAR2 agonist) over 24 hours further indicatedfunctional receptor activity, in that magnesium significantly inhibitedproliferation of all cell types (Table 2, p<0.05).

TABLE 2 Fluorescence (nm) Cell Line Vehicle Antagonist LAN2 420 150 MCF7800 550 SKBr3 330 160 Results represent triplicate studies performedwith alamar blue on LAN2 neuroblastoma, MCF-7 breast cancer, and SKBr3breast cancer cells examining the growth influence of 100 mM Mg⁺⁺ in thepresence of glycine over 24 hours.

Further, it was found an affinity-purified preparation of polyclonalantibody against the N-terminal region (SEQ ID NO:5) of human NMDAR1(PANN1) when administered at 1:50 (<<10 ng, <<0.075 pmol antibody) or1:10 (<<50 ng, <<0.38 pmol antibody) could decrease the proliferation ofsmall cell lung cancer H345 cells in culture by about 6% and 25%,respectively, compared to controls (Table 3).

TABLE 3 Fluorescence (nm) Treatment 24 Hours 48 Hours 72 Hours Control,1:50 723.5 ± 3.8 789.2 ± 13.1 610.7 ± 4.1 PANN1, 1:50 678.0 ± 2.8**784.3 ± 7.1 605.8 ± 3.8 Control, 1:10 811.3 ± 5.5 927.2 ± 8.2 596.5 ±3.7 PANN1, 1:10 617.0 ± 2.9** 665.5 ± 7.4** 507.0 ± 2.2** **p < 0.001.Fluorescence due to alamar blue is a linear representation ofproliferation.

Moreover, treatment with NMDAR1 antagonist, MK801, was found to decreaseproliferation of SKBr3 and MCF-7 breast cancer cells. For example, aconcentration of 0.9 mM reduced proliferation of SKBr3 cells at 72 hoursby about 80% (Table 4).

TABLE 4 Proliferation of SKBr3 Cells Percent Fluorescence Compared toControl MK801 (μM) 24 Hours 48 Hours 72 Hours 100 92.29 ± 6.82 91.74 ±2.94 95.06 ± 1.22 200 89.47 ± 5.77 89.35 ± 2.83 93.24 ± 1.68 300 84.39 ±5.01 83.79 ± 2.71 88.97 ± 2.04 400 75.17 ± 4.67 70.62 ± 2.34 77.08 ±2.78 500 65.50 ± 3.74 59.07 ± 2.49 67.02 ± 5.13 600 58.06 ± 3.00 50.59 ±2.34 52.57 ± 2.28 700 51.97 ± 2.77 43.84 ± 3.41 44.88 ± 3.40 800 43.27 ±2.50 33.77 ± 4.19 33.91 ± 4.35 900 35.73 ± 2.66 25.48 ± 5.92 25.21 ±5.96

Likewise, NMDAR1 antagonist memantine decreased the proliferation ofMCF-7 and SKBr3 breast cancer cells. For example, concentration of 0.3mM was found to reduce proliferation of MCF-7 cells at 48 and 72 hoursby about 80% (Table 5).

TABLE 5 Proliferation of MCF-7 Cells Percent Memantine FluorescenceCompared to Control (μM) 24 Hours 48 Hours 72 Hours 100 92.86 ± 6.0889.98 ± 5.30 88.48 ± 4.02 200 73.51 ± 5.64 49.78 ± 6.85 46.62 ± 7.04 30048.81 ± 5.18 22.33 ± 5.21 20.02 ± 4.97 400 39.62 ± 5.21 15.79 ± 4.7914.38 ± 3.92 500 38.40 ± 2.98 14.60 ± 3.21 13.40 ± 3.29 600 39.22 ± 4.1315.10 ± 7.28 13.66 ± 6.62 700 32.80 ± 2.19 12.51 ± 2.35 11.89 ± 2.62 80032.82 ± 2.44 12.34 ± 2.01 11.82 ± 2.25 900 32.23 ± 2.13 12.03 ± 2.1511.57 ± 2.41

NMDAR antagonist activity on the viability of small-cell cancer cellswas also analyzed. Significant effects (p<0.01) on cell viability bydifferent antagonists (reductions to 10% control or increases to 140%control) were found at all three incubation times of 24, 48, and 72hours. The changes induced following 48 hours and 72 hours of incubationwere similar, and greater than those found following 24 hours ofincubation. Effects were also different for the different cell lines.Memantine and MK-801 produced dramatic decreases (p<0.0001) in cellviability (FIGS. 1 and 2), with IC₅₀s at 48 hours of 80-130 μM forNCI-H345 and NCI-H82, 400 μM for NCI-H146, and 800 μM for DMS-53 cellswith memantine; and of 300 μM for NCI-H345 and NCI-H82, 650 μM forNCI-H146, and >800 μM for DMS-53 with MK-801. Both Ifenprodil and Ro25-6981 also decreased cell viability (FIG. 3) with respective IC₅₀s at48 hours of ˜150 and ˜200 μM for both NCI-H345 and NCI-H82 cells, but areduction no greater than 40% with NCI-H146, and smaller decreases withDMS-53 with Ifenprodil, and no clear effects at the concentration rangeused with Ro 25-698. The effects of the glycine site binders were onlyexamined on NCI-H345 and NCI-H82. At 48 hours, L-701,324 produced anincrease in cell viability to approximately 140% and 120% of control forNCI-H345 and NCI-H82, respectively. At 48 hours, L-701, 252 alsoproduced an increase to approximately 120% of control for NCI-H345cells, but no clear effect over the concentration range for NCI-H82cells. Differences were observed in these studies between different celllines and these differences probably reflect either a concentrationrange of NMDARs or a differing dependence of the cells on functionalNMDARs for growth. However, effects were greatest on the one variantcell line (and one classical cell line) and this variant cell linerepresents recurrent and drug-resistant disease. Moreover, it is ofinterest that preventing glycine from binding to NMDAR2 receptors seemedto improve cell survival.

The ability of an NMDAR1 antibody to inhibit tumor cell growth was alsodemonstrated. An affinity-purified preparation of PENN1 (anti-NMDAR1)rabbit polyclonal antibodies was incubated with small cell lung cancerNCI-H345 cells for 24 hours at 1:50 (<<10 ng, <75 fmol antibody) or 1:10(<<50 ng, <0.375 fmol antibody). The results of this analysis indicatedthat the antibody significantly (P<0.001) decreased the proliferation ofsmall cell lung cancer NCI-H345 cells in culture by about 6% and 25%,respectively, compared to controls (Table 6).

TABLE 6 Treatment (24 Hour) Fluorescence of Alamar Blue Control, 1:50723.5 ± 3.8 AntiNMDAR1, 1:50  678.0 ± 2.8** Control, 1:10 811.3 ± 5.5AntiNMDAR1, 1:10  617.0 ± 2.9** *Significant at p < 0.001 compared withcontrol

These data showed an importance of NMDAR1 receptors in the proliferationof this SCLC cell line, and demonstrated the availability of theantigenic site on the receptors for antibody binding, wherein suchbinding disrupts signaling through these channels. Additionally, thedata indicates that increased doses of the PENN1 antibody or similarantibodies, especially directing cell cytotoxicity in vitro and in vivo,could kill most, or all such cancer cells. Because antigenic sequencesof human NMDAR1 protein and mouse NMDAR1 protein are identical, mice aresuitable models to study the effects of anti-NMDAR1s on tumors in vivo.

Accordingly, NMDAR antagonism was monitored in vivo in a mouse tumormodel. The treatment of established tumor xenografts in nu/nu mice withMK-801, at the dose indicated herein, produced no noticeable behavioraleffects in the animals and had no apparent effect on their well-being asexemplified by body weight in treated animals paralleling that forcontrols. However, as shown in FIG. 4, MK-801 produced highlysignificant (p<0.001) reductions in the rate of tumor growth, to almost60% that of controls, over the course of the study. This effect ongrowth was evident for even the lowest dose used, i.e., 0.1 mg/Kg bodyweight.

These effects further support the findings that neuroendocrine tumorcells in culture and neuroendocrine tumors express both NMDAR1 plusNMDAR2 (e.g., NMDAR2B and NMDAR2A) receptors, and that the function ofthese receptors is to influence the growth and viability of thesecells/tumors. Accordingly, the present invention is also a method fordecreasing or inhibiting the proliferation of a neuroendocrine tumorcell (e.g., in vitro or in vivo cells or cell lines of neuroendocrinetumors disclosed herein) using an agent which inhibits or decreases theactivity or expression of an NMDA glutamate receptor. As used herein, anagent which inhibits or decreases the activity of an NMDA glutamatereceptor is intended to include binding agents (e.g., antibodies orpeptide aptamers as disclosed herein), antagonists, partial antagonistsand the like which have the effect of blocking the ability of the NMDAglutamate receptor to bind modulatory compounds (e.g., glutamate,glycine, or polyamines) or block the channel pore thereby decreasing theactivity of the NMDA glutamate receptor. An agent which inhibits ordecreases the expression of an NMDA glutamate receptor is intended toinclude small molecules or nucleic acids which hybridize with nucleicacids coding for an NMDA glutamate receptor and inhibit or decrease theexpression thereof.

Antagonists useful in accordance with the methods of the presentinvention include antagonists which act at the glutamate binding site ofNMDA glutamate receptors. Exemplary antagonists of this type include,but are not limited to, L-glutamate derivatives,(R)-2-amino-5-phosphonopentanoate,(R)-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid,(±)-cis-4-(4-phenylbenzoyl)piperazine-2,3-dicarboxylic acid and thelike.

Antagonism of the glycine binding site will also antagonize NMDAreceptor function. A series of high affinity antagonists have beendeveloped for this binding site on the NMDA receptor and include, butare not limited to, L-701,324; L-689,560; and GV96771A, which displaceglycine binding with affinities below 10 nM.

The effects of polyamine site antagonists are specific for channelscontaining the NR2B subunit. Hence, a polyamine site antagonist whichwould be useful as an NR2B antagonist for this site includes, but is notlimited to, Ro 25-6981.

Channel blockers refer to compounds that bind to the pore of the NMDAreceptor channel and block transport of cations (e.g., Na⁺, K⁺ and Ca²⁺ions). NMDA receptor channel blockers useful in accordance with themethods of the present invention include, e.g., magnesium, dizocilpine,phencyclidine, ketamine, memantine, tiletamine, budipine, flupirtine,1-[1-(2-thienyl)cyclohexyl]piperidine (TCP),(+)-(3S,4S)-7-hydroxy-delta6-tetrahydrocannabinol-1,1-dimethylheptyl(HU211) and MK801.

Glutamate-related agonists are further contemplated. High levels ofglutamate-related agonists in the presence of glycine were found to becytotoxic to neuroendocrine tumor cells in vitro. Suitableglutamate-related agonists for use in accordance with the methods of thepresent invention, include, but are not limited to, homoquinolinic acid,NPG and ACDA.

In particular embodiments of the instant methods, an agent whichinhibits or reduces the activity of an NMDA glutamate receptor is abinding agent which binds to at least a portion of the N-terminalextracellular domain of NMDAR1 or NMDAR2. Such binding agents includeantibodies, antibody fragments and peptide aptamers as disclosed supra.

Agents useful for decreasing the expression on an NMDA glutamatereceptor include agents having a sequence complementary to at least partof NMDA glutamate receptor nucleic acid sequence. Without being limitedby theory, the inhibition of expression by the agent is achieved throughselective hybridization with NMDA glutamate receptor DNA or mRNA therebyimpeding any steps in the replication, transcription, splicing ortranslation of a NMDA glutamate receptor nucleic acid. Examples ofagents that can be used decrease expression of an NMDA glutamatereceptor include, but are not limited to, antisense oligonucleotides,ribozymes, nucleic acids molecules that promote triple helix formation,and siRNAs or co-repression of a target gene by introducing a homologousgene fragment into the cell that harbors the target gene.

A sequence is complementary when it hybridizes to its target sequenceunder high stringency, i.e., conditions for hybridization and washingunder which nucleotide sequences, which are at least 60 percent(preferably greater than about 70, 80, or 90 percent) identical to eachother, typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art, and can be found, forexample, in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6. Another example of stringent hybridizationconditions is hybridization of the nucleotide sequences in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by 0.2×SSC, 0.1%SDS at 50-65° C. Another example of stringent hybridization conditionsis hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 55° C. Afurther example of stringent hybridization conditions is hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. Alternatively, astringent hybridization condition is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C. Another alternative example ofstringent hybridization condition is 0.5 M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

Antisense nucleotide sequences that can be used to decrease or inhibitthe expression of an NMDA glutamate receptor include nucleotidesequences that are complementary to the nucleotide sequences including,but are not limited to, GENBANK accession numbers NM_(—)000832,NM_(—)021569, NM_(—)007327, NM_(—)000833, NM_(—)000834, NM_(—)000835,and NM_(—)000836 or paralogs or orthologs, or portions thereof. Further,an antisense nucleotide sequence can be designed that is specific for analternatively spliced variant of an NMDA glutamate receptor by directingthe antisense nucleotide sequence to nucleic acid sequences specific tothe variant of interest. A particularly suitable NMDA glutamatereceptor-specific antisense oligonucleotide is disclosed in Weyermann etal. ((2004) J. Control. Release. 100(3):411-23).

Those skilled in the art can appreciate that it is not necessary thatthe antisense nucleotide sequence be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to hybridize to its target and reduceproduction of NMDA glutamate receptor protein (e.g., by at least about40%, 50%, 60%, 70%, 80%, 90%, 95% or more). As is known in the art, ahigher degree of sequence similarity is generally required for shortantisense nucleotide sequences, whereas a greater degree of mismatchedbases will be tolerated by longer antisense nucleotide sequences.

Alternatively stated, antisense nucleotide sequences should have atleast about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequencesimilarity with the complement of the NMDA glutamate receptor codingsequences to reduce the level of NMDA glutamate receptor production.

The length of the antisense nucleotide sequence is not critical as longas it binds selectively to the intended location and reducestranscription and/or translation of the target sequence. In general, theantisense nucleotide sequence will be from about eight, ten or twelvenucleotides in length up to about 20, 30, 50, 60 or 70 nucleotides, orlonger, in length. Further, it is contemplated that peptide

In another embodiment, RNA interference (RNAi) is used to modulate NMDAglutamate receptor expression. RNAi has proven successful in humancells, including human embryonic kidney and HeLa cells (see, e.g.,Elbashir, et al. (2001) Nature 411:494-8). The mechanism by which RNAiachieves gene silencing has been reviewed in Sharp, et al. (2001) GenesDev 15:485-490; and Hammond, et al. (2001) Nature Rev. Gen. 2:110-119).Accordingly, NMDA glutamate receptor expression can be inhibited byintroducing into or generating within a cell (i.e., transgenicexpression) an siRNA molecule corresponding to an NMDA glutamatereceptor-encoding nucleic acid or fragment thereof. In variousembodiments, such a method can entail the direct administration of thesiRNA or siRNA-like molecule into a cell, or use of the vector-basedmethods. In one embodiment, the siRNA molecule is less than about 30nucleotides in length. In a further embodiment, the siRNA molecule isabout 21-23 nucleotides in length. In another embodiment, an siRNAmolecule is a 19-21 bp duplex, each strand having a two nucleotide 3′overhang. Kits for production of dsRNA for use in RNAi are availablecommercially, e.g., from New England Biolabs, Inc. and Ambion Inc.(Austin, Tex., USA). Methods of transfection of dsRNA or plasmidsengineered to make dsRNA are routine in the art.

Silencing effects similar to those produced by RNAi have been reportedin mammalian cells with transfection of a mRNA-cDNA hybrid construct(Lin, et al. (2001) Biochem. Biophys. Res. Commun. 281:639-44),providing yet another strategy for silencing a coding sequence ofinterest.

In a further embodiment, the agent can further be a ribozyme. Ribozymecatalysis has primarily been observed as part of sequence-specificcleavage/ligation reactions involving nucleic acids (Joyce (1989) Nature338:217). For example, U.S. Pat. No. 5,354,855 reports that certainribozymes can act as endonucleases with sequence specificities greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon, et al. (1991) Proc. Natl. Acad. Sci. USA88:10591; Sarver, et al. (1990) Science 247:1222; Sioud, et al. (1992)J. Mol. Biol. 223:831).

As will be appreciated by the skilled artisan, stable oligonucleotideanalogs can also be used for inhibiting or reducing expression of anNMDA glutamate receptor. Such analogs can have the negatively chargedsugar-phosphate backbone replaced by a polypeptide backbone leading toenhanced stability and the formation of stronger hybrids withcomplementary RNA and DNA. Likewise 2′-sugar modifications are alsocontemplated.

Neuroendocrine tumor cell proliferation is decreased or inhibited bycontacting tumor cells with an effective amount of one or more of theagents disclosed herein which inhibit or decrease the activity orexpression of an NMDA glutamate receptor. Desirably, the agent decreasesthe neuroendocrine tumor cell proliferation by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100% as compared to the same cell in theabsence of the agent; an effect which can be determined in accordancewith standard methods for measuring rates of cell proliferation.

By inhibiting or decreasing neuroendocrine tumor cell proliferation,this method of the present invention will be useful as a research toolfor studying the signaling pathways involved in the growth anddevelopment of neuroendocrine tumors and for preventing or treatingneuroendocrine tumors in vivo.

Therefore, the present invention is also a method for preventing ortreating a neuroendocrine cancer in a subject by administering to asubject having or at risk of having a neuroendocrine cancer an effectiveamount of an agent which inhibits or decreases the activity orexpression of an NMDA glutamate receptor. Prevention or treatmenttypically involves the steps of first identifying a subject having or atrisk of having a neuroendocrine cancer. Individuals having cancergenerally refers to subjects who have been diagnosed with neuroendocrinecancer and require treatment, whereas individuals at risk of having aneuroendocrine cancer may have a family history of such a cancer orexhibit one or more signs or symptoms associated with such a cancer andrequire prevention of the same. Once such a subject is identified using,for example, standard clinical practices, the subject is administered apharmaceutical composition containing an effective amount of an agentwhich inhibits or decreases the activity or expression of an NMDAglutamate receptor (e.g., agents disclosed supra). In most cases, thesubject being treated will be a human being, but treatment ofagricultural animals, e.g., livestock and poultry, and companionanimals, e.g., dogs, cats and horses, is expressly covered herein.

In particular embodiments of the instant method, an agent that inhibitsor decreases the activity of an NMDA glutamate receptor is a bindingagent which binds to at least a portion of the N-terminal extracellulardomain of NMDAR1 or NMDAR2. Such binding agents include antibodies,antibody fragments and peptide aptamers as disclosed supra.Advantageously, binding agents such as antibodies and antibody fragmentswill not generally cross the blood-brain barrier and bind to NMDAglutamate receptors located in central neurons. Therefore, side-effectsassociated with inactivation of neuronal NMDA glutamate receptors willbe minimized.

The selection of the dosage or effective amount of the agent is thatwhich has the desired outcome of reducing or reversing at least one signor symptom of neuroendocrine cancer. For example, depending on thecancer, some of the general signs or symptoms can include a tumor,increased pain perception, weakness, abdominal pain, anemia, pneumonia,a cough, and spitting of blood.

For therapeutic use, the agent is generally formulated with apharmaceutically acceptable vehicle, such as water, buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like) or suitable mixtures thereof. The optimumconcentration of the active ingredient(s) in the chosen vehicle can bedetermined empirically, according to procedures well-known to medicinalchemists. As used herein, pharmaceutically acceptable vehicle includesany solvent, dispersion medium, and the like which may be appropriatefor the desired route of administration of the pharmaceuticalpreparation. The use of such vehicle for pharmaceutically activesubstances is known in the art. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in Remington:The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20thed. Lippingcott Williams & Wilkins: Philadelphia, Pa., 2000.

A pharmaceutical composition containing an agent (e.g., a binding agentor antisense oligonucleotide) which blocks NMDA glutamate receptoractivity or expression and a pharmaceutically acceptable vehicle can beused alone or in combination with other well-established agents usefulfor preventing or treating cancer. Whether delivered alone or incombination with other agents, the pharmaceutical composition of thepresent invention can be delivered via various routes and to varioussites in a mammalian, particularly human, body to achieve a particulareffect. One skilled in the art will recognize that, although more thanone route can be used for administration, a particular route can providea more immediate and more effective reaction than another route. Forexample, pulmonary delivery may be advantageously used over subcutaneousdelivery for the treatment of SCLC. Local or systemic delivery can beaccomplished by application or instillation of the formulation into bodycavities; inhalation or insufflation of an aerosol; or by parenteralintroduction, including intramuscular, intravenous, intraportal,intrahepatic, peritoneal, subcutaneous, or intradermal administration.

Those of ordinary skill in the art can readily optimize effective dosesand co-administration regimens as determined by good medical practiceand the clinical condition of the individual patient. Regardless of themanner of administration, it can be appreciated that the actualpreferred amounts of active agent in a specific case will vary accordingto the particular formulation and the route of administration. Thespecific dose for a particular patient depends on age, body weight,general state of health, on diet, on the timing and route ofadministration, on the rate of excretion, and on medicaments used incombination and the severity of the particular disorder to which thetherapy is applied. Dosages for a given subject can be determined usingconventional considerations, e.g., by customary comparison of thedifferential activities of the selected agent and of a known agent, suchas by means of an appropriate conventional pharmacological protocol. Byway of illustration, dosing of an antibody raised against at least aportion of the N-terminal extracellular domain of NMDAR1 or NMDAR2 canbe based upon dosing of antibodies such as ERBITUX® (Cetuximab orIMC-C225) and HERCEPTIN® (Trastuzumab) currently used in the treatmentof breast cancer and colorectal cancer, respectively.

In addition to being directly useful in the prevention and treatment ofneuroendocrine cancers, the binding agents of the present invention arealso useful as cell-surface targeting moieties as they have been shownto specifically bind to neuroendocrine tumor cells and not normal cells.A cell-surface targeting moiety is defined as an agent whichspecifically targets a chemotherapeutic, therapeutic, radiotherapeuticor in situ imaging agent to a neuroendocrine cancer cell.

Chemotherapy and therapeutic anticancer agents which will be used inconjunction with a neuroendocrine cancer targeting moiety of theinvention include, cytotoxic agents such as Taxol, Cytochalasin B,Gramicidin D, Ethidium Bromide, Emetine, Mitomycin, Etoposide,Tenoposide, Vincristine, Vinblastine, camptothecin (CPT), Colchicin,Doxorubicin, Daunorubicin, Mitoxantrone, Mithramycin, Actinomycin D,1-Dehydrotestosterone, Glucocorticoids, Procaine, Tetracaine, Lidocaine,Propranolol, blocked ricin (Lynch, et al. (1997) J. Clin. Oncol.15(2):723-34) and Puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa, chlorambucil, melphalan, carmustine, lomustine,cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, cis-dichlorodiamine platinum (II), cisplatin), anthracyclines (e.g.,daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin,bleomycin, mithramycin, and anthramycin), anti-mitotic agents (e.g.,vincristine and vinblastine) and selective apoptotic agents such asAPTOSYN® (exisulind), PANZEM™ (2-methoxyestradiol), and VELCADE®(bortezomib). Radiotherapeutic agents which can be targeted toneuroendocrine tumors via binding agents are well-known in the art. See,e.g., Ballangrud, et al. (2004) Clin. Cancer Res. 10(13):4489-97. NMDAglutamate receptor binding agents can also advantageously be used totarget NMDA glutamate receptor agonists or antagonists to NMDA glutamatereceptors located on neuroendocrine tumor cells as such binding agentsshould not cross the blood-brain barrier.

The use of an NMDA glutamate receptor as a neuroendocrine tumor antigenfor vaccinating a subject having or at risk of having a neuroendocrinetumor is also expressly contemplated herein. A vaccine can include anisolated NMDA glutamate receptor, or an antigenic fragment thereof, anda pharmaceutically acceptable vehicle. Using art-established methods, anisolated NMDA glutamate receptor protein or antigenic fragment can berecombinantly-produced, chemically-synthesized, or isolated from cellswhich naturally express an NMDA glutamate receptor. Alternatively,nucleic acid encoding an NMDA glutamate receptor protein or antigenicfragment can be part of a viral-based vaccine. One or more amino acids,not corresponding to the original protein sequence can be added to theamino or carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the protein toanother peptide, to a large carrier protein, or to a support which canenhance the immunological response to the vaccine. Amino acids that areuseful for these purposes include: tyrosine, lysine, glutamic acid,aspartic acid, cysteine and derivatives thereof. Alternative proteinmodification techniques can be used, e.g., NH₂-acetylation orCOOH-terminal amidation, to provide additional means for coupling orfusing the protein/peptide to another protein or peptide molecule or toa support. Active immunization can be induced by administering one ormore antigenic and/or immunogenic epitopes of an NMDA glutamate receptorprotein as a component of a vaccine. Vaccination can be performedorally, nasally or parenterally in amounts sufficient to enable therecipient to generate protective antibodies against this biologicallyfunctional region, prophylactically or therapeutically. In addition to apharmaceutically acceptable vehicle, the NMDA glutamate receptorantigen, or antigenic fragment thereof, can be co-administered with anadjuvant to enhance the immunological response to the vaccine. During etal. ((2000) Science 287(5457):1453-60) teach a particularly suitableadeno-associated virus (AAV) vaccine for generating autoantibodies toNMDAR1. The single-dose vaccine disclosed was well-tolerated and able toproduce a neuroprotective activity in rats. Such a vaccine will beuseful as an immuno-based approach to tumor eradication and prevention.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Cell Culture

Breast cancer (i.e., MCF-7 and SKBR3) and SCLC (i.e., classical humanSCLC cell lines NCI-H345, DMS-53, NCI-H146, and the variant SCLC cellline, NCI-H82,) cells were maintained at 37° C. in an atmosphere of 5%CO₂ using Dulbecco's Minimal Essential Medium (DMEM; 0.8 mM Mg²⁺, 0.4 mMglycine; Sigma, St. Louis, Mo.) or RPMI 1640 (Mediatech, Inc., Herndon,Va.) supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin.Every 3-4 days, cells received fresh DMEM or were subcultured using0.06% trypsin with 0.02% EDTA. Cell densities were at 10⁵ to 5×10⁵/ml.

MCF-7 and SKBR3 are art-established cell cultures for analyzing thepathophysiology of breast cancer and preclinical analysis of drugefficacy (Hanauske (2004) Oncology (Huntingt). 18(13 Suppl 8):66-9). Inparticular, studies using MCF-7 cultures have correlated well with invivo therapeutic studies. For example, Johnson, et al. ((1997) SeminOncol. 24(1 Suppl 3):S22-5) teach that paclitaxel and 5-fluorouracilhave additive cytotoxicity in MCF-7 cell lines and is an active,well-tolerated regimen in metastatic breast cancer.

Example 2 RNA Isolation, RT-PCR and Northern Blot Analysis

RNA Isolation. Poly(A)⁺ RNA was isolated from cells using oligo(dT)cellulose chromatography in accordance with well-established methods(Badley, et al. (1988) Biotechniques 5:114-116).

RT-PCR. Poly(A)⁺ RNA (2 μg) from breast cancer or SCLC cells wasdenatured at 70° C. for 10 minutes and chilled on ice. First strand cDNAsynthesis was carried with a SUPERSCRIPT™ preamplification system(GIBCO-BRL®, Gaithersburg, Md.), using an oligo(dT) primer and 1 μL (200U) of SUPERSCRIPT™ II reverse transcriptase (PROMEGA®, Madison, Wis.).The reverse transcriptase product was directly used as a template forthe PCR reaction. PCR was carried out using GENEAMP® PCR reagents(PERKIN ELMER™, Foster City, Calif.) in a thermocycler (EASY CYCLER™Series, ERICOMP, San Diego, Calif.). The templates were initiallydenaturated at 97° C. for 8 minutes and amplified for 30 cycles underthe following conditions: denaturation at 95° C. for 30 seconds;annealing at 58° C. for 1.5 minutes; and extension at 72° C. for 1.5minutes. After the cycling reaction was complete, an additionalextension step was carried out at 72° C. for 10 minutes. Primers used inthe amplification of the NMDAR1 receptor included F1, 5′-ATC TAC TCG GACAAG AGC ATC C-3′ (SEQ ID NO:1, corresponding to nucleotides 369 to 417of the sequence set forth in GENBANK Accession No. L05666); R1, 5′-AGCTCT TTC GCC TCC ATC AG-3′ (SEQ ID NO:2, corresponding to complimentnucleotides 658 to 639 of GENBANK Accession No. L05666); F2,5′-AAG TATGCG GAT GGG GTG ACT-3′ (SEQ ID NO:3, nucleotides 1002 to 1022 of GENBANKAccession No. L05666); R2, 5′-CAA AAG CCG TAG CAA CAC TGA-3′ (SEQ IDNO:4, nucleotides 1393 to 1373 of GENBANK Accession No. L05666). NMDAR2Bprimers included forward primer 1,5′-TCA AGG ATG CCC ACG AGA AAG-3′ (SEQID NO:6, corresponding to nucleotide residues 358 to 378 of GENBANKAccession No. NM_(—)000834); reverse primer 1, 5′-GTG GCT TCT TCC TTGTGA CAG-3 (SEQ ID NO:7, corresponding to nucleotide residues 922 to 902of GENBANK Accession No. NM_(—)000834); forward primer 2, 5′-CCA AAG AGCATC ATC ACC C-3′ (SEQ ID NO:8, corresponding to nucleotide residues 441to 459 of GENBANK Accession No. NM_(—)000834); reverse primer 2, 5′-TGTAGC CAT AGC CAG TCA G-3′ (SEQ ID NO:9, corresponding to nucleotideresidues 972 to 954 of GENBANK Accession No. NM_(—)000834). NMDAR2Aprimers included forward primer, 5′-TCC AAT AGT GCC CTG CTA AG-3′ (SEQID NO:10) and reverse primer, 5′-tgc caa cat acc cag tag gc-3′ (SEQ IDNO:11), which amplify a 220 base pair fragment corresponding tonucleotides 4780-4999 of GENBANK Accession No. NM_(—)000833.2 (humanGRIN2A). These primers were synthesized by a commercial source. The PCRproducts were purified by washing once with an equal volume ofchloroform and examined on 2% agarose gels. DNase-free RNase andRNase-free DNase (GIBCO/BRL) were employed in some reactions to validatethat the products of PCR did not result from initial DNA contamination.

Sequencing. To confirm that the amplicons encoded the correct NMDARprotein, they were cloned into PCR™ vector and transformed into ONESHOTT™ Competent Cells using a TA CLONING® kit (INVITROGEN™, San Diego,Calif.). At least two clones of each amplicon were selected fordouble-stranded cDNA sequencing with a TAQ DYEDEOXY™ Terminator CycleSequencing Kit (APPLIED BIOSYSTEMS™, Foster City, Calif.). F1, F2, R1and R2 primers in combination with universal primers M13 Forward, M13reverse, and T7 were used as sequencing primers. The protocol for DNAamplification was modified as follows: 97° C., 2 minutes; 25 cycles of95° C., 30 seconds; 58° C., 1.5 minutes; and 72° C., 1.5 minutes with a72° C. extension for 10 minutes. Amplicons were purified and sequencingwas performed using Model 373 DNA Sequencer (APPLIED BIOSYSTEMS™, FosterCity, Calif.). Upon BLAST analysis, it was determined that the ampliconfor NMDAR1 coded for a segment of the N-terminal extracellular domain ofNMDAR1 which was nearly identical in sequence to NMDAR1 from humanneurons.

Northern Blot Analysis. Northern blot analysis was performed with 10 μgof poly(A)⁺ RNA from breast cancer and SCLC cells. RNA was denatured andfractionated on a 1.2% formaldehyde agarose gel. Separated products weretransferred to nitrocellulose membranes (NITRO-PURE™; MSI, Westboro,Mass.) with 10×SSC transfer buffer, vacuum-dried for 2 hours, andprehybridized at 42° C. for 4 hours. PCR amplicons were radiolabeledwith [³²P]dCTP (3000 Ci/mmol; DUPONT®/NEN, Boston, Mass.) using theDECAPRIME II™ DNA labeling kit (Ambion, Inc., Austin, Tex.) and used tohybridize with the membrane at 42° C. for 18 hours. The membrane waswashed twice at room temperature, first in a 2×SSC solution containing0.1% SDS and then in a 0.1×SSC solution containing 0.1% SDS. Themembrane was subsequently washed twice under stringent conditions of 55°C. in 0.1×SSC containing 0.1×SSC and exposed to X-ray film for 5 to 7days.

PANN1 is a rabbit polyclonal antiserum produced against an N-terminalsegment of NMDAR1. Antibodies from this antiserum were first isolated byaffinity chromatography. Commercially available NMDAR1 antiserum wasobtained from Cell Signaling, and NMDAR2 antiserum was purchased fromSanta Cruz (NMDAe2, Santa Cruz Biochemicals, Santa Cruz, Calif.). Thelatter antiserum recognizes all NMDAR2 subunits. After analysis withanti-NMDAR antibodies, the blots were stripped and incubated with GAPDH(Chemicon), or scanned with anti-β-actin (SIGMA), to ensure equalprotein loading.

Example 3 Neutral Red Assay

Breast cancer and SCLC cells were subcultured into 24-well plates(CORNING®, Corning, N.Y.). After 24 hours, the growth medium wasaspirated and replaced with growth medium containing L-glutamic acid(Sigma, St. Louis, Mo.) at concentrations of 0, 1, or 10 mM, orN-phthalamoyl-L-glutamic acid (NPG; Research Biochemicals Inc., Natick,Mass.) at concentrations of 0, 0.1 or 1 mM. Following 48 hours ofincubation at 37° C., the experimental medium was removed and replacedwith DMEM containing 40 μg/mL neutral red (Sigma, St. Louis, Mo.). Aftera two hour incubation, the neutral red was aspirated and the cellmonolayers carefully washed with phosphate-buffered saline (PBS).Incorporated dye was extracted from cells with 50% ethanol/1% aceticacid. The absorbance of recovered dye was determined at 540 nm orfluorescence measured at excitation of 530 nm and emission 650 nm.

Example 4 Immunocytochemistry

Samples, i.e., cancer cell lines or cancer tissue isolated frompatients, were trypsinized and plated into chamber slides (Nunc,Naperville, Ill.). Subsequently, the samples were washed with PBS,blocked with 1.5% goat serum for 30 minutes, and fixed in acetone for 2minutes. Samples were washed with PBS and stained for about 1 hour atroom temperature with either rabbit polyclonal antiserum raised againsta NMDAR1 peptide of SEQ ID NO:5 or rabbit polyclonal antiserum raisedagainst NMDAR2abcd (Santa Cruz Biochemicals, Santa Cruz, Calif.) in PBScontaining 10% normal goat serum (GIBCO-BRL®, Gaithersburg, Md.).Samples were then washed with PBS and incubated for 30 minutes withbiotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, Calif.).Samples were subsequently washed with PBS and incubated withavidin-peroxidase complex for 30 minutes. Visualization of theantigen-antibody complex was performed by the peroxidase oxidation of3,3′-diaminobenzidine. A negative control incubated with 1.5% goat-seruminstead of primary antibody confirmed that staining was not as a resultof non-specific reactivity of the secondary antibody.

Example 5 Cell Growth/Viability Assay

Cultures of NCI-H345, NCI-H146, DMS-53 cells (classical cell lines), andNCI-H82 cells (variant cell line) were treated with 0.05% trypsin,washed in phosphate buffered saline (PBS)/glycine and plated onto96-well plates at 10⁴ cells/well in medium for 24 hours to provide timefor rectifying possible damage to membrane proteins from thetrypsinization. The wells were washed and incubated with PBS/glycinecontaining albumin (1.25 mg/mL) in the presence or absence of differentconcentrations (25 μM-800 μM) of either the NMDAR 1 antagonists,Memantine or MK-801; or the NMDAR2B antagonists Ifenprodil and Ro25-6981 (binders to the polyamine site), or the NMDAR2B antagonistsL-701,252 and L-701,324 (binders to the glycine site), or vehicle; andAlamar Blue (1:10 dilution following manufacturer's recommendation).Fluorescent readings were taken at periods representing 24, 48, and 72hours of incubation with antagonist using a Synergy HT Multi-DetectionMicroplate Reader and excitation/emission wavelengths of 530 nm and 590nm. Cell viability was evaluated as % vehicle control at thecorresponding incubation time.

Rabbit polyclonal anti-NMDAR1 antibodies (PANN1) were generated againstan N-terminal extracellular peptide domain(Met-Ser-Ile-Tyr-Ser-Asp-Lys-Ser-Ile-His (SEQ ID NO:5)) of the humanreceptor, in the form of the peptide amide coupled throughglutaraldehyde to bovine thyroglobulin as antigen, and use of Fruend'scomplete and incomplete adjuvant (Friedmann, et al. (1994) Br. J. Cancer69:260-263; Keegan, et al. (2002) Mol. Cancer. Therapeutics.1:1153-1159). This peptide domain is represented in that portion of themRNA message sequenced by RT-PCR. For studies on cell proliferation, theIgGs of this antiserum were first isolated by precipitation with 50%saturated ammonium sulfate, and antibodies purified from theseimmunoglobulins by affinity chromatography using a column containing thepeptide component of the above antigen coupled to SEPHAROSE (Friedmann,et al. (1994) supra; Keegan, et al. (2002) supra). Two dilutions (1/10and 1/50) of an affinity-purified PANN1 antibody preparation in PBS/0.5%BSA (<<375 fmol Ab and <<75 fmol Ab) were incubated with cells in 1640medium for 24 hours at 37° C., and cell growth compared with cellstreated with liquid vehicle using the Alamar Blue procedure.

Example 6 Treatment of SCLC Tumor Xenografts in Mice

Human tumor xenografts of small cell lung cancer (SCLC) cell lineNCI-H345 were raised in nu/nu mice, and the influence of the NMDAR1receptor antagonist dizocilpine maleate (MK-801) then examined onestablished tumor growth measured by multiplying depth, width, andlength determined using a micrometer. Tumor growth in a control group ofanimals receiving i.p. PBS vehicle (n=4) was compared to tumor growth inanimals (n=4) receiving dizocilpine maleate (MK-801) over 10 days.Animals received an escalating single dose of this NMDAR1 antagonistfrom 0.1 mg/Kg body weight each day for days 0-2, then to a single doseof 0.2 mg/Kg body weight each day for days 3-6, then to a single dose of0.3 mg/Kg body weight each day for days 7-8. Finally two daily doses of0.3 mg/Kg body weight were given for days 8-10.

Results were analyzed by Analysis of Variance and theStudent-Neuman-Kuels test. Longitudinal growth data was evaluated usingrepeated measures ANOVA. Significant was determined to be present forp<0.05.

1. A method for diagnosing small cell lung cancer (SCLC) or breastcancer comprising isolating a lung tissue sample or breast tissue samplefrom a subject, detecting the presence of at least one NMDA glutamatereceptor protein in the lung tissue sample or breast tissue sample, anddiagnosing the subject as having SCLC or breast cancer, respectively,when the presence of the at least one NMDA glutamate receptor protein isdetected.